Project Summary/Abstract The ultimate goal of this proposal is to address a fundamental gap in knowledge on the role of the cell cycle inhibitor p16 in regulating pro-tumorigenic metabolism. The results from these studies could have a significant impact on the treatment of melanoma patients, of which ~30-40% have downregulation or deletion of p16. This research plan focuses on experimentally and mechanistically determining the role of p16 loss in pro-tumorigenic nucleotide metabolism and whether this pathway can be targeted in p16-low melanomas alone or in combination with mutant BRAF inhibitors to obtain a sustained therapeutic response. The proposed studies are based on preliminary findings that loss of p16 expression upregulates the newly-identified ATR-mTORC1 signaling axis to increase nucleotide metabolism through the pentose phosphate pathway, and modulation of this pathway is a metabolic vulnerability for p16-low cancer cells. In line with these data, we will explore two overarching scientific aims: 1) to mechanistically dissect the ATR-mTORC1 pathway downstream of p16 loss in melanomagenesis and determine the contribution of pro-tumorigenic nucleotide metabolism to the observed phenotypes; and 2) to elucidate whether targeting the ATR-mTORC1 pathway in combination is synergistic in p16-low melanomas alone or in combination with mutant BRAF inhibitors. The completion of the scientific aims of this proposal will not only provide new mechanistic insights into the interplay between metabolism and the cell cycle during tumorigenesis, but will also establish targeting the novel ATR-mTORC1 axis as a strategy to improve therapeutic outcome for melanoma patients with low p16 expression. The proposed research is of high impact because the mechanistic underpinning of these pathways has the potential to transform the management of melanomas with low p16. As p16 is altered in ~50% of all human cancers, these studies will have far-reaching implications for identifying metabolic vulnerabilities and developing future cancer therapeutic strategies for a wide range of patients.